Polyinosinic-polycytidylic acid has therapeutic effects against cerebral ischemia/reperfusion injury through the downregulation of TLR4 signaling via TLR3.

Recent reports have shown that preconditioning with the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)) protects against cerebral ischemia/reperfusion (I/R) injury. However, it is unclear whether poly(I:C) treatment after cerebral I/R injury is also effective. We used mouse/rat middle cerebral artery occlusion and cell oxygen-glucose deprivation models to evaluate the therapeutic effects and mechanisms of poly(I:C) treatment. Poly(I:C) was i.p. injected 3 h after ischemia (treatment group). Cerebral infarct volumes and brain edemas were significantly reduced, and neurologic scores were significantly increased. TNF-α and IL-1ß levels were markedly decreased, whereas IFN-ß levels were greatly increased, in the ischemic brain tissues, cerebral spinal fluid, and serum. Injuries to hippocampal neurons and mitochondria were greatly reduced. The numbers of TUNEL-positive and Fluoro-Jade B(+) cells also decreased significantly in the ischemic brain tissues. Poly(I:C) treatment increased the levels of Hsp27, Hsp70, and Bcl2 and decreased the level of Bax in the ischemic brain tissues. Moreover, poly(I:C) treatment attenuated the levels of TNF-α and IL-1ß in serum and cerebral spinal fluid of mice stimulated by LPS. However, the protective effects of poly(I:C) against cerebral ischemia were abolished in TLR3(-/-) and TLR4(-/-)mice. Poly(I:C) downregulated TLR4 signaling via TLR3. Poly(I:C) treatment exhibited obvious protective effects 14 d after ischemia and was also effective in the rat permanent middle cerebral artery occlusion model. The results suggest that poly(I:C) exerts therapeutic effects against cerebral I/R injury through the downregulation of TLR4 signaling via TLR3. Poly(I:C) is a promising new drug candidate for the treatment of cerebral infarcts.

Toll-like receptor 2/4 heterodimer mediates inflammatory injury in intracerebral hemorrhage.

OBJECTIVE: Inflammatory injury plays a critical role in intracerebral hemorrhage (ICH)-induced secondary brain injury. However, the upstream events that initiate inflammatory responses following ICH remain elusive. Our previous studies suggested that Toll-like receptor 4 (TLR4) may be the upstream signal that triggers inflammatory injury in ICH. In addition, recent clinical findings indicated that both TLR2 and TLR4 may participate in ICH-induced brain injury. However, it is unclear how TLR2 functions in ICH-induced inflammatory injury and how TLR2 interacts with TLR4. METHODS: The role of TLR2 and TLR2/TLR4 heterodimerization in ICH-induced inflammatory injury was investigated in both in vivo and in vitro models of ICH. RESULTS: TLR2 mediated ICH-induced inflammatory injury, which forms a heterodimer with TLR4 in both in vivo and in vitro models of ICH. Hemoglobin (Hb), but not other blood components, triggered inflammatory injury in ICH via assembly of TLR2/TLR4 heterodimers. MyD88 (myeloid differentiation primary response gene 88), but not TRIF (Toll/IR-1 domain-containing adaptor protein inducing interferon-beta), was required for ICH-induced TLR2/TLR4 heterodimerization. Mutation of MyD88 Arg196 abolished the TLR2/TLR4 heterodimerization. INTERPRETATION: Our results suggest that a novel TLR2/TLR4 heterodimer induced by Hb initiates inflammatory injury in ICH. Interfering with the assembly of the TLR2/TLR4 heterodimer may be a novel target for developing effective treatment of ICH.

Why ligand cross-reactivity is high within peptide recognition domain families? A case study on human c-Src SH3 domain.

Many important protein-protein interactions in eukaryotic signaling networks are mediated by peptide recognition domains (PRDs), which bind short linear sequence motifs in other proteins. However, high ligand cross-reactivity is observed within most PRD families, rendering a broad specificity for the family members. In the present study, we attempt to explore the molecular mechanism and physicochemical origin of PRD cross-reactivity. In the procedure, a structure-based method called atomic cross-nonbonded interaction analysis (ACNIA) is described to extract atomic-level nonbonded interaction information at domain-peptide interface and to correlate the information with peptide affinity based on a set of structure-solved, affinity-known protein-peptide complex samples compiled from numerous literatures and databases. The ACNIA-derived affinity predictor is tested rigorously with statistical validation approach, which is also demonstrated to be capable of perceiving slight structural change in the interface using three distinct panels of SH3-binding peptide data. Subsequently, with help of the affinity predictor we adopt the human c-Src SH3 domain, one of the most sophisticated PRDs, as a paradigm to investigate the ligand cross-reactivity within SH3 family. It is found that most of the family members have only few non-essential residue differences in their peptide-binding pockets, and thus exhibit a similar peptide recognition profile and high cross-reactivity. The cross-reactivity is even shared by different subclasses of SH3 domains. The findings suggest that inherent binding specificity is not the only factor to select appropriate binders for specific SH3 domains, and other aspects such as cellular context and the rest of the SH3-containing proteins may play important roles in reducing their ligand cross-reactivity.

Heme activates TLR4-mediated inflammatory injury via MyD88/TRIF signaling pathway in intracerebral hemorrhage.

BACKGROUND: Inflammatory injury plays a critical role in intracerebral hemorrhage (ICH)-induced neurological deficits; however, the signaling pathways are not apparent by which the upstream cellular events trigger innate immune and inflammatory responses that contribute to neurological impairments. Toll-like receptor 4 (TLR4) plays a role in inflammatory damage caused by brain disorders. METHODS: In this study, we investigate the role of TLR4 signaling in ICH-induced inflammation. In the ICH model, a significant upregulation of TLR4 expression in reactive microglia has been demonstrated using real-time RT-PCR. Activation of microglia was detected by immunohistochemistry, cytokines were measured by ELISA, MyD88, TRIF and NF-κB were measured by Western blot and EMSA, animal behavior was evaluated by animal behavioristics. RESULTS: Compared to WT mice, TLR4(-/-) mice had restrained ICH-induced brain damage showing in reduced cerebral edema and lower neurological deficit scores. Quantification of cytokines including IL-6, TNF-α and IL-1ß and assessment of macrophage infiltration in perihematoma tissues from TLR4(-/-), MyD88(-/-) and TRIF(-/-) mice showed attenuated inflammatory damage after ICH. TLR4(-/-) mice also exhibited reduced MyD88 and TRIF expression which was accompanied by decreased NF-κB activity. This suggests that after ICH both MyD88 and TRIF pathways might be involved in TLR4-mediated inflammatory injury possibly via NF-κB activation. Exogenous hemin administration significantly increased TLR4 expression and microglial activation in cultures and also exacerbated brain injury in WT mice but not in TLR4(-/-) mice. Anti-TLR4 antibody administration suppressed hemin-induced microglial activation in cultures and in the mice model of ICH. CONCLUSIONS: Our findings suggest that heme potentiates microglial activation via TLR4, in turn inducing NF-κB activation via the MyD88/TRIF signaling pathway, and ultimately increasing cytokine expression and inflammatory injury in ICH. Targeting TLR4 signaling may be a promising therapeutic strategy for ICH.

[Effect of C5a on expression of thrombomodulin in endothelial cells in vitro].

OBJECTIVE: To investigate the effect of C5a on the expression of thrombomodulin (TM) in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs cultured in vitro were stimulated with C5a for 8, 12, 16, 20 hours in a concentration of 200 microg/L, and also with different concentrations of 100, 200, 300 microg/L for 12 hours respectively. The levels of both mRNA and protein expression of TM was detected by real-time polymerase chain reaction (PCR) and Western blotting respectively, and the dose and time dependent effects of C5a on the expression of TM were evaluated. RESULTS: C5a down-regulated the TM expression at both protein and mRNA level. The down-regulation was time-dependent [(93.11+/- 1.57) x10(-2) , (71.05+/-3.39)x10(-2), (65.48+/-4.28)x10(-2), (62.69+/-4.03)x10(-2)at protein level and (301.71+/-80.40)x10(-6), (38.29+/-20.24)x10(-6), (8.82+/-2.66)x10(-6), (7.05+/-0.80)x10(-6) at mRNA level, all P<0.05] and dose-dependent [(113.25+/-3.97)x10(-2), (80.18+/-2.56)x10(-2), (73.22+/- 4.36)x10(-2) at protein level and (401.77+/-20.46)x10(-6), (31.12+/-3.51)x10(-6), (18.19+/-1.46)x10(-6) at mRNA level , all P<0.05]. When concentration of C5a at 300 microg/L was used to stimulate HUVECs for longer than 12 hours, the lowering of TM at protein level was slowed down obviously. And concentration of C5a at 200 microg/L was used to stimulate HUVECs for 12 hours, the lowering of TM at mRNA level was slowed down obviously. CONCLUSION: C5a can depress the gene expression of TM, and then affect the protein's translation. By this means, C5a can lead to hypercoagulability and inflammatory injuries in sepsis.

Role of Toll-like receptor 4/NF-kappaB pathway in monocyte-endothelial adhesion induced by low shear stress and ox-LDL.

TLR4 plays an important role in atherosclerosis, but little is known about the precise mechanism. Herein, we investigated the role of TLR4/NF-kappaB signaling pathway in monocyte-endothelial adhesion induced by low shear stress and Ox-LDL. We found that low shear stress up-regulated TLR4 expression in endothelial cells, and that ox-LDL exerted an obvious synergistic action as revealed by RT-PCR and Western blotting analysis. Low shear stress also significantly up-regulated IL-8 expression in endothelial cells. Meanwhile, NF-kappaB activity and the adhesion force of monocytes were increased, and there was a synergetic action of ox-LDL. However, following transfection with a functional mutant of TLR4 (C3H/HeJ, TLR4 Dicd) or addition of anti-human TLR4 mAb, IL-8 expression was obviously decreased, NF-kappaB activity in cells remarkably inhibited, and the adhesion force of monocyte significantly reduced. Nevertheless, anti-human TLR2 mAb had no similar effects. These findings suggest that TLR4 may be involved in the early stages of atherosclerosis, associating ox-LDL, inflammation/infection, and low shear stress. Therefore, TLR4 is expected to be a new target for preventing and treating atherosclerosis.

The auto-inhibitory state of Rho guanine nucleotide exchange factor ARHGEF5/TIM can be relieved by targeting its SH3 domain with rationally designed peptide aptamers.

The short isoform of Rho guanine nucleotide exchange factor ARHGEF5 is known as TIM, which plays diverse roles in, for example, tumorigenesis, neuronal development and Src-induced podosome formation through the activation of its substrates, the Rho family of GTPases. The activation is auto-inhibited by a putative helix N-terminal to the DH domain of TIM, which is stabilized by the intramolecular interaction of C-terminal SH3 domain with a poly-proline sequence between the putative helix and the DH domain. In this study, we systematically investigated the structural basis, energetic landscape and biological implication underlying TIM auto-inhibition by using atomistic molecular dynamics simulations and binding free energy analysis. The computational study revealed that the binding of SH3 domain to poly-proline sequence is the prerequisite for the stabilization of TIM auto-inhibition. Thus, it is suggested that targeting SH3 domain with competitors of the poly-proline sequence would be a promising strategy to relieve the auto-inhibitory state of TIM. In this consideration, we rationally designed a number of peptide aptamers for competitively inhibiting the SH3 domain based on modeled TIM structure and computationally generated data. Peptide binding test and guanine nucleotide exchange analysis solidified that these designed peptides can both bind to the SH3 domain potently and activate TIM-catalyzed RhoA exchange reaction effectively. Interestingly, a positive correlation between the peptide affinity and induced exchange activity was observed. In addition, separate mutation of three conserved residues Pro49, Pro52 and Lys54 - they are required for peptide recognition by SH3 domain -- in a designed peptide to Ala would completely abolish the capability of this peptide activating TIM. All these come together to suggest an intrinsic relationship between peptide binding to SH3 domain and the activation of TIM.

[Change in Toll-like receptor 4 expression on interstitial macrophages in acute lung injury].

OBJECTIVE: To observe changes in the level of Toll-like receptor 4 (TLR4) mRNA and the expression of TLR4 protein in interstitial macrophages in serious thoracic injury. METHODS: A rat model of severe thoracic trauma was reproduced,then interstitial macrophages were isolated and collected by enzymatic digestion before and after trauma cultured. TLR4 mRNA and the expression of TLR4 protein were measured by Northern and Western blotting before trauma and 2, 4, 8, 16, 24 hours after trauma. RESULTS: A stable and reliable severe thoracic trauma model was successfully reproduced with 400 kPa impact on the up-right chest of rat by a multiple-function collision apparatus. The expression was up-regulated by trauma and the highest expression of interstitial macrophages occurred 8, 16 hours after the trauma, and returned to normal range at 24 hours. CONCLUSION: The up-regulation of TLR4 might participate in the pathological process of excessive inflammation after trauma to the lung.

Expression of the HPV18E2 gene in cervical cancer and premalignant lesions and its clinical significance.

This study aims to observe the expression of the HPV18E2 gene in cervical cancer and premalignant lesions and to investigate its clinical significance. The expression of the HPV18E2 gene in the cervical tissues obtained from 38 women with cervical lesions was detected using the RT-PCR method. The pathological changes were graded based on cervical intraepithelial neoplasia (CIN) criteria. The HPV18E2 gene was expressed mainly in cervical premalignant lesions, 60 % in Grade I CIN, 33.3 % in Grade II CIN, and 28.6 % in Grade III CIN. No expression was detected in cervical cancer and chronic cervical inflammation. This study suggests that peptides vaccine targeting the HPV18E2 protein may disrupt and prohibit the progress of diseases induced by HPV 18 infection (i.e., CIN and cervical cancer).

Proteome-wide inference of human endophilin 1-binding peptides.

Human endophilin 1 (hEndo1) is a multifunctional protein that was found to bind a wide spectrum of prolinerich endocytic proteins through its Src homology 3 (SH3) domain. In order to elucidate the unknown biological functions of hEndo1, it is essential to find out the cytoplasmic components that hEndo1 recognizes and binds. However, it is too time-consuming and expensive to synthesize all peptide candidates found in the human proteome and to perform hEndo1 SH3-peptide affinity assay to identify the hEndo1-binding partners. In the present work, we describe a structure/ sequence-hybrid approach to perform proteome-wide inference of human hEndo1-binding peptides using the information gained from both the primary sequence of affinity-known peptides and the interaction profile involved in hEndo1 SH3-peptide complex three-dimensional structures. Modeling results show that (i) different residue positions contribute distinctly to peptide affinity and specificity; P-1, P2 and P4 are most important, P1 and P3 are also effective, and P-3, P-2, P0, P5 and P6 are relatively insignificant, (ii) the consensus core PXXP motif is necessary but not sufficient for determining high affinity of peptides, and some other positions must be also essential in the hEndo1 SH3-peptide binding, and (iii) the alternating arrangement of polar and nonpolar amino acids along peptide sequence is critical for the high specificity of peptide recognition by hEndo1 SH3 domain. In addition, we also find that the residue type at a specific position of hEndo1-binding peptides is not stringently invariable; amino acids that possess similar polarity could replace each other without substantial influence on peptide affinity. In this way, hEndo1 presents a broad specificity in the peptide ligands that it binds.

Toward an improved discrimination of outer membrane proteins using a sequence-based approach.

This article offers a novel sequence-based approach to discriminate outer membrane proteins (OMPs). The first step is to use a new representation approach, factor analysis scales of generalized amino acid information (FASGAI) representing hydrophobicity, alpha and turn propensities, bulky properties, compositional characteristics, local flexibility and electronic properties, etc., to characterize sequences of OMPs and non-OMPs. The subsequent data is then transformed into a uniform matrix by the auto cross covariance (ACC). The second step is to develop discrimination predictors of OMPs from non-OMPs using a support vector machine (SVM). The SVM predictors thus successfully produce a high Matthews correlation coefficient (MCC) of 0.916 on 208 OMPs from non-OMPs including 206 α-helical membrane proteins and 673 globular proteins by a fivefold cross validation test. Meanwhile, overall MCC values of 0.923 and 0.930 are obtained for the discrimination OMPs from the α-helical membrane proteins and the globular proteins, respectively. The results demonstrate that the FASGAI-ACC-SVM combination approach shows great prospect of application in the field of bioinformatics or proteomics studies.

Reduced cerebral ischemia-reperfusion injury in Toll-like receptor 4 deficient mice.

Inflammatory reaction plays an important role in cerebral ischemia-reperfusion injury, however, its mechanism is still unclear. Our study aims to explore the function of Toll-like receptor 4 (TLR4) in the process of cerebral ischemia-reperfusion. We made middle cerebral artery ischemia-reperfusion model in mice with line embolism method. Compared with C3H/OuJ mice, scores of cerebral water content, cerebral infarct size and neurologic impairment in C3H/Hej mice were obviously lower after 6 h ischemia and 24 h reperfusion. Light microscopic and electron microscopic results showed that cerebral ischemia-reperfusion injury in C3H/Hej mice was less serious than that in C3H/OuJ mice. TNF-alpha and IL-6 contents in C3H/HeJ mice were obviously lower than that in C3H/OuJ mice with ELISA. The results showed that TLR4 participates in the process of cerebral ischemia-reperfusion injury probably through decrease of inflammatory cytokines. TLR4 may become a new target for prevention of cerebral ischemia-reperfusion injury. Our study suggests that TLR4 is one of the mechanisms of cerebral ischemia-reperfusion injury besides its important role in innate immunity.

Novel TLR4-antagonizing peptides inhibit LPS-induced release of inflammatory mediators by monocytes.

Toll-like receptor 4 (TLR4) has become a new target for combating Gram-negative bacterium-induced sepsis. In this study, we screened peptides that can interact with TLR4 from a random 16-peptide library using yeast two-hybrid system and performed functional identification for the obtained peptides. We got two positive clones out of 1.28x10(7) transformants. The peptides were sequenced and synthesized. Protein sequence comparison confirmed that the two peptides had no homologous proteins. The two peptides were found to significantly inhibit LPS-induced NF-kappaB activation in HEK-293 cells that were transfected with TLR4 cDNA, LPS-induced IkappaBalpha (IkappaB alpha) phosphorylation and NF-kappaB activation in monocytes, and release of IL-1, IL-6, and TNF-alpha by monocytes. We further confirmed that the No. 9 peptide could bind to TLR4 extracellular domain, but the No. 24 peptide could not, suggesting that two novel peptides were identified as the antagonists of TLR4, which significantly inhibited the effects of endotoxin in vitro. The No. 9 peptide may function through binding to TLR4 extracellular domain. Our findings suggest a promising countermeasure against Gram-negative bacterium-induced sepsis.

[An experimental study of inhibiting effect of decoy oligodeoxynucleotides on the gene expression of collagen in NIH3T3 cell].

OBJECTIVE: To investigate the effect of activator protein-1 (AP-1) decoy-oligodeoxynucleotides (Decoy-ODNs) on the expression of fibroblast alpha2 type I collagen, so as to explore the gene therapy of pathologic scar. METHODS: Decoy-ODNs targeting AP-1 were designed and synthesized. NIH3T3 cells were transfected by cationic liposomes. The distribution of Decoy-ODNs in the cells was investigated. The inhibiting effects of Decoy-ODNs on AP-1 were determined by electrophoretic mobility shift assay (EMSA). And the effects of Decoy-ODNs on the collagen synthesis in the cells were analyzed by RT-PCR. RESULTS: AP-1 Decoy-ODNs could competitively inhibit the AP-1 in vitro activity. Cationic liposomes could play roles by effectively transfecting Decoy-ODNs into the plasma and nucleus. The mRNA expression of fibroblast alpha2 type I collagen decreased evidently after 24 hours of Decoy-ODNs action. CONCLUSION: Decoy-ODNs could inhibit the mRNA expression of fibroblast alpha2 type I collagen by antagonizing AP-1.